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I. L thuyt 1.1. Gii thiu khi qut v Nit protein Tt c cc dng nit c trong c th hay trong cc m c gi l nit tng s. Nit c trong thnh phn axit amin ca protein l N protein. Nit khng c trong thnh phn protein nh cc mui m v c, axit nitric, cc axit amin t do, ure v cc dn xut ca ure, cc alcaloit, cc baz purin v pyrimidinl cc nit phi protein. Nit tng s = nit protein + nit phi protein 1.2. Phng php ly mu Ly mu sn phm kim nghim l khu u tin v rt quan trng trong cng tc kim nghim. Vic ly mu ng quy cch s gp phn cho kt qu kim nghim v x l sau ny ng n. V trong thc t, ch ly mt lng mu rt nh kim nghim m kt qu li c dng nh gi mt cch khch quan cht lng sn phm c khi lng rt ln. V vy khi ly mu chng ta cn thc hin cc quy nh

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di y: + Mu thc phm phi c tnh cht i din cho c l hng thc phm ng nht. + Trc khi ly mu cn kim tra tnh ng nht ca l hng, xem xt cc giy t km theo, i chiu nhn trn bao b, ring cc sn phm c bao b khng cn nguyn vn (rch, thng, v, mt nhn,) phn chia s cn li thnh l hng ng nht. + i vi sn phm th rn: Cn ch n s khng ng u v kch thc ca sn phm, phi ly c sn phm c kch thc ln v kch thc b. Thng ta tin hnh chia im ly mu tu theo hnh dng ca n v cha sn phm. + i vi sn phm va th rn, va th lng khng ng nht khi ly mu, ly cc v tr khc nhau nhng phi ly c phn rn, c phn lng theo ng t l ca chng trong sn phm. + i vi sn phm dng st ng nht, khuy k v ly mu u cc v tr khc nhau. + i vi sn phm c ng trong hp, chai, l hoc bao gi trong tith ly mu c bao gi. 1.3. Cc phng php phn tch Protein 1.3.1 nh lng Protein bng phng php Lowry + Nguyn tc: Da vo phn ng mu ca protein v thuc th Folin, cng mu ca dung dch t l thun vi nng protein, v da vo ng chun ca protein, c th tnh c hm lng protein ca mu nghin cu. 1.3.2 nh lng Protein bng phng php Coomassie Brilliant Blue G 250 + Nguyn tc: Phng php ny da vo s thay i mu xy ra khi Coomassie Brilliant Blue G 250 lin kt vi protein trong dung dch axit. Dng proton ho ca thuc nhum Coomassie Blue c mu da cam. Thuc nhum lin kt cht ch vi cc protein, tng tc vi c nhm k nc v cc nhm mang in tch dng trn

phn t protein. Trong mi trng ca cc gc mang in tch dng, s proton ho khng xy ra v c mu xanh xut hin. 1.3.3 nh lng Protein bng phng php quang ph + Nguyn tc: Pht hin v o protein: Phng php n gin nht o nng protein trong dung dch l hp th tia cc tm ca n. Nu protein tinh sch th nng tuyt i ca n c tnh theo gi tr c o. Nu protein khng tinh sch th nng ca protein tng s c tnh tng i t hp ph. 1.3.4 nh lng Protein bng phng php Dumas + Nguyn tc: Phng php t chy Dumas xc nh protein th. Quy trnh dng mt thit b l in un nng mu phn tch ln n 600oC trong mt l phn ng c bt kn vi s hin din ca oxy. Hm lng nit ca kh t sau c o bng cch dng my d dn nhit. Mi ln xc nh ch cn khong 2 pht. 1.3.5 nh lng N - Protein bng my Kjeldahl 1.3.5.1 Nguyn tc ca qu trnh phn tch Mu c v c ho bng H2SO4 nhit cao v c cht xc tc. Cc phn ng ca qu trnh v c ho mu xy ra nh sau: 2H2SO4 2 H2O + 2SO2 + O2 Oxy to thnh trong phn ng li oxy ho cc nguyn t khc. Cc phn t cha nit di tc dng ca H2SO4 to thnh NH3. Cc protein b thu phn thnh axit amin. Cacbon v hydro ca axit amin to thnh CO2 v H2O, cn nit c gii phng di dng NH3 kt hp vi axit H2SO4 d to thnh (NH4)2SO4 tan trong dung dch. 2NH3 + H2SO4 (NH4)2SO4 Cc nguyn t khong khc nh P, K, Ca, Mg,chuyn thnh dng oxit: P2O5, K2O, CaO, MgOtuy tn ti trong dung dch mu nhng khng nh hng n kt qu phn tch. ui amoniac ra khi dung dch bng NaOH.

(NH4)2SO4 + 2NaOH = Na2SO4 + H2O + 2NH3 NH3 bay ra cng vi nc sang bnh hng. Bnh hng c cha H3BO3 do NH3 bay ra s tc dng ngay vi H3BO3 theo phn ng: 2NH3 + 2H2O + 4H3BO3 = (NH4)2B4O7 + 7H2O Lng (NH4)2B4O7 c xc nh thng qua vic chun bng HCl 0,25N cho n khi dung dch chuyn sang mu hng nht v khng b mt mu. 1.3.5.2. Nhng yu t c th nh hng n kt qu phn tch + Nng HCl 0,25N khng m bo chnh xc + Ly mu khng i din + Cn mu khng chnh xc + Ngun nc b nhim N 1.3.5.3 Kh nng ng dng ca phng php Kjeldahl Phng php ny c kh nng ng dng cho hu ht cc loi mu: + Mu thc phm: thu hi sn, nc mm, cc loi u, thc n nui tm c, rau qu + t, nc thi + B men bia, bnh du u nnh, bnh du cao su 1.4. Nguyn tc hot ng ca my Kjeldahl Mu c a vo bn t (h thng v c ho mu) thng qua cc ng Kjeldahl Sau khi v c ho mu xong, ton b mu + ng Kjeldahl c a qua h thng chng ct NH3 ton b sn phm c a qua thit b chun Kt Qu. II. Thc Hnh 2.1. Qui trnh Phn tch mu u phng: Bc 1: Chun b mu -Mu ht c nghin mn -Sy mu 80oC cho n khi t trng lng khng i Bc 2: V c ho mu

-Cn 0,3 1 g mu kh c nghin mn. -Cho mu vo tn y ca ng Kjeldahl (chu khng dnh trn thnh ng) -Cho 5g hn hp xc tc K2SO4 v CuSO4 theo t l 10:1 -Dng pipet ht 15ml H2SO4 m c (d=1,84) cho vo ng v c ho mu c cha hn hp trn. -Lp ng trn vo h thng v c ho mu. -Vic v c ho mu hon ton khi ton b dung dch trong ng v c ho mu c mu xanh trong sut. Bc 3: Chng ct mu -Lp ng v c ho mu vo b chng ct, lp bnh hng cha 10-60ml H3BO3 4% v 7 git dung dch ch th mu. -Khi ng my ct. -Qu trnh chng ct tin hnh khong 390 giy th ton b kh NH3 chuyn sang bnh hng. -Khi NH3 c ct hon ton, h bnh hng xung, dng tia nc ct nh trng sch axit dnh u ng lm lnh. Bc 4: Chun Bc 5: Tnh kt qu Ta c 1ml HCl 0,25N tng ng vi 0,0035g N. Thng qua s ml HCl 0,25N, ngi ta bit c s lng axit boric kt hp vi NH3, t bit lng NH3 gii phng t mu. N tng (%) = (VHCl * 0,35)/ P mu Trong : V tnh bng ml P tnh bng g Protein tng (%) = (VHCl * 0,35* 6,25)/ P mu 2.2. Mt s thit b v ho cht s dng 2.2.1 Thit b: - T sy mu

- My nghin mu - Cn phn tch 4 s - My phn tch m t ng Kjeldahl - Thit b chun - My ct nc 2 ln 2.2.2 Ho cht: - Hn hp K2SO4 v CuSO4 theo t l 10:1 - Nc ct 2 ln - NaOH 32% - H2SO4 m c - HCl 0,24N - Acid Boric 4% - Cht ch th mu: cch pha: + Ho tan 0,264g methyl trong 250 ml C2H5OH (cn tuyt i) + Ho tan 1,28g Bromocresol xanh l cy trong 50ml C2H5OH (cn tuyt i) +Trn u 2 dung dch trn, b sung 96ml HCl 0,25N. nh mc n 1000ml bng C2H5OH.
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Ni dung thc tp chuyn gen vo t bo thc vt(03-02-2009) Phng php xc nh m (21-01-2009) ELISA nng cao(21-01-2009) Nguyen Minh Nam Research Institute for Biotechnology and Environment Nong Lam University- Ho Chi Minh CityVietnam. Tel: (84 8) 8972262-112 Mobile: (84)904972804 Fax: (848) 8972262-103 Email: mnam-az @hcmuaf.edu.vn/ saophuongnam05 @gmail.com/saophuongnam05 @yahoo.com Thit k: Qun tr mng- HNL 2007